Phytochemistry (v.66, #3)

Author Index (III).

Update in bioinformatics by Marcela Beatriz Treviño Santa Cruz; Dominique Genoud; Jean-Pierre Métraux; Thierry Genoud (267-276).
The properties of a digital modeling of signal transduction networks and the potential benefits of this method for creating a computer database of plant signaling events within a ‘digital plant’ project are presented.The process of signal integration, which contributes to the regulation of multiple cellular activities, can be described in a digital language by a set of connected digital operations. In this article we delineate the basic concepts of cell signalling in the context of a logical description of information processing. Newly described instances of signal integration in plants are given as examples. The different advantages, limitations and predictive aspects of the digital modeling of signal transduction networks, as well as the minimal architecture of a computer database for plant signalling networks are discussed.
Keywords: Cell signalling database; Signal transduction networks; Digital networks; Digital simulation; Cross-talks; Phytochrome;

Starter substrate specificities of wild-type and mutant polyketide synthases from Rutaceae by Richard Lukačin; Stephan Schreiner; Katrin Silber; Ulrich Matern (277-284).
Chalcone and acridone synthases catalyse similar condensations of 4-coumaroyl-CoA and N-methylanthraniloyl-CoA, respectively, with three malonyl-CoAs, and the enzyme polypeptides show 75–85% sequence homology. Mutant chalcone synthases were generated from Ruta CHS1 with the aim to confer acridone synthase activity. Homology modeling and docking studies suggested that a Phe267Val and further conformational changes in the periphery of the polypeptide backbone are essentially required.Chalcone synthases (CHSs) and acridone synthases (ACSs) belong to the superfamily of type III polyketide synthases (PKSs) and condense the starter substrate 4-coumaroyl-CoA or N-methylanthraniloyl-CoA with three malonyl-CoAs to produce flavonoids and acridone alkaloids, respectively. ACSs which have been cloned exclusively from Ruta graveolens share about 75–85% polypeptide sequence homology with CHSs from other plant families, while 90% similarity was observed with CHSs from Rutaceae, i.e., R. graveolens, Citrus sinensis and Dictamnus albus. CHSs cloned from many plants do not accept N-methylanthraniloyl-CoA as a starter substrate, whereas ACSs were shown to possess some side activity with 4-coumaroyl-CoA. The transformation of an ACS to a functional CHS with 10% residual ACS activity was accomplished previously by substitution of three amino acids through the corresponding residues from Ruta-CHS1 (Ser132Thr, Ala133Ser and Val265Phe). Therefore, the reverse triple mutation of Ruta-CHS1 (mutant R2) was generated, which affected only insignificantly the CHS activity and did not confer ACS activity. However, competitive inhibition of CHS activity by N-methylanthraniloyl-CoA was observed for the mutant in contrast to wild-type CHSs. Homology modeling of ACS2 with docking of 1,3-dihydroxy-N-methylacridone suggested that the starter substrates for CHS or ACS reaction are placed in different topographies in the active site pocket. Additional site specific substitutions (Asp205Pro/Thr206Asp/His207Ala or Arg60Thr and Val100Ala/Gly218Ala, respectively) diminished the CHS activity to 75–50% of the wild-type CHS1 without promoting ACS activity. The results suggest that conformational changes in the periphery beyond the active site cavity volumes determine the product formation by ACSs vs. CHSs in R. graveolens. It is likely that ACS has evolved from CHS, but the sole enlargement of the active site pocket as in CHS1 mutant R2 is insufficient to explain this process.
Keywords: Ruta graveolens L; Dictamnus albus; Rutaceaee; Acridone synthase; Chalcone synthase; Site-directed mutagenesis;

A geraniol-synthase gene from Cinnamomum tenuipilum by Tao Yang; Jing Li; Hao-Xin Wang; Ying Zeng (285-293).
A cDNA clone from Cinnamomum tenuipilum was isolated, functionally expressed in Escherichia coli and thereby identified as a single copy gene coding for a geraniol synthase. The CtGES is more abundantly expressed in leaves of a geraniol chemotype than in those of either linalool or farnesol chemotypes.Geraniol may accumulate up to 86–98% of the leaf essential oils in geraniol chemotypes of the evergreen camphor tree Cinnamomum tenuipilum. A similarity-based cloning strategy yielded a cDNA clone that appeared to encode a terpene synthase and which could be phylogenetically grouped within the angiosperm monoterpene synthase/subfamily. After its expression in Escherichia coli and enzyme assay with prenyl diphosphates as substrates, the enzyme encoded by the putative C. tenuipilum monoterpene synthase gene was shown to specifically convert geranyl diphosphate to geraniol as a single product by GC–MS analysis. Biochemical characterization of the partially purified recombinant protein revealed a strong dependency for Mg2+ and Mn2+, and an apparent Michaelis constant of 55.8 μM for geranyl diphosphate. Thus, a new member of the monoterpene synthase family was identified and designated as CtGES. The genome contains a single copy of CtGES gene. Expression of CtGES was exclusively observed in the geraniol chemotype of C. tenuipilum. Furthermore, in situ hybridization analysis demonstrated that CtGES mRNA was localized in the oil cells of the leaves.
Keywords: Cinnamomum tenuipilum; Lauraceae; Camphor tree; Functional expression; Chemotypes; Essential oil; CtGES gene; Geraniol synthase; Monoterpene synthases;

The metabolism of deuterium labeled geraniol 5 in grape mesocarp of Vitis vinifera L. cv. Scheurebe was studied by in vivo-feeding experiments. Stereoselective reduction to (S)-citronellol 9, stereoselective biosynthesis of the potent odorant cis-(2S,4R)-rose oxide 11 and E/Z-isomerization to nerol 7 could be demonstrated.The metabolism of deuterium labeled geraniol in grape mesocarp of Vitis vinifera L. cv. Scheurebe was studied by in vivo-feeding experiments. Stereoselective reduction to (S)-citronellol, E/Z-isomerization to nerol, oxidation to neral/geranial and glycosylation of the corresponding monoterpene alcohols could be demonstrated. Time course studies including the determination of conversion rates revealed that the activity of these secondary transformations of monoterpenes is dependent on the ripening stage and can be distinguished from the development of the primary monoterpene synthase activities by the sharp increase at the end of the ripening period. The stereoselective biosynthesis of the potent odorant cis-(2S,4R)-rose oxide from labeled geraniol in grape berry mesocarp is demonstrated as well. Since (S)-citronellol is the precursor of cis-(2S,4R)-rose oxide it can be concluded that especially the last part of the ripening period is important for the generation of this potent odorant. This finding confirms the conclusion that a higher concentration of flavor compounds could be established in the berries by leaving the fruit on the vine for extended periods.
Keywords: Vitis vinifera; Monoterpenes; Rose oxide; Citronellol; Biosynthesis; Enantioselective multidimensional gas chromatography–mass spectrometry (enantio-MDGC–MS); Stir bar sorptive extraction (SBSE);

The biosynthesis of the monoterpenes terpinolene and myrcene and the sesquiterpene β-caryophyllene in roots and leaves of two carrot varieties were investigated.The biosynthesis of the monoterpenes terpinolene and myrcene and the sesquiterpene β-caryophyllene in roots and leaves of two carrot varieties (Daucus carota L. cultivars Bolero and Kazan) were investigated by in vivo feeding experiments with [5,5-2H2]-mevalonic acid lactone (d2-MVL) and [5,5-2H2]-1-deoxy-d-xylulose (d2-DOX). The volatiles of the tissues were extracted by stir bar sorptive extraction and analyzed using thermal desorption–multidimensional gas chromatography–mass spectrometry. The experiments demonstrate independent de novo-biosynthesis of terpenoids in carrot roots and in carrot leaves. In both plant tissues monoterpenes are biosynthesized exclusively via the 1-deoxy-d-xylulose/2-C-methyl-d-erythritol-4-phosphate (DOXP/MEP) pathway, whereas sesquiterpenes are generated by the classical mevalonic acid pathway as well as by the DOXP/MEP route. A more detailed investigation of carrot root tissues revealed that the biosynthesis of terpenes is mainly localized in the phloem. Nevertheless, in xylem a de novo-biosynthesis of terpenes was detectable as well, even in the absence of oil ducts in this tissue.
Keywords: Daucus carota L.; Umbelliferae; 1-Deoxy-d-xylulose; Mevalonic acid; Stir bar sorptive extraction; Thermal desorption–multidimensional gas chromatography–mass spectrometry;

Visible light-induced oxidation of unsaturated components of cutins: a significant process during the senescence of higher plants by Jean-François Rontani; Adélaı¨de Rabourdin; Franck Pinot; Sylvie Kandel; Claude Aubert (313-321).
Visible light-induced oxidation of 18-hydroxyoleic acid (a cutin component) was observed in senescent leaves of parsley. The photoproducts thus formed were then detected in different natural samples.9-Hydroperoxy-18-hydroxyoctadec-10(trans)-enoic and 10-hydroperoxy-18-hydroxyoctadec-8(trans)-enoic acids deriving from type II (i.e. involving 1O2) photooxidation of 18-hydroxyoleic acid were detected after visible light-induced senescence experiments carried out with Petroselinum sativum and subsequent cutin depolymerisation. These results showed that in senescent plants, where the 1O2 formation rate exceeds the quenching capacity of the photoprotective system, 1O2 can migrate outside the chloroplasts and affect the unsaturated components of cutins. Significant amounts of 9,18-dihydroxyoctadec-10(trans)-enoic and 10,18-dihydroxyoctadec-8(trans)-enoic acids resulting from the reduction of these photoproducts of 18-hydroxyoleic acid were also detected in different natural samples. These results well support the significance of the photooxidation of the unsaturated components of higher plant cutins in the natural environment.
Keywords: Petroselinum sativum; Cutins; Higher plants; Senescence; 18-Hydroxyoleic acid; Photooxidation; Singlet oxygen;

Metabolic flux analysis in complex isotopolog space. Recycling of glucose in tobacco plants by Christian Ettenhuber; Tanja Radykewicz; Waltraud Kofer; Hans-Ulrich Koop; Adelbert Bacher; Wolfgang Eisenreich (323-335).
Tobacco plants grown on agar were supplied with [U-13C6]glucose via the root system. Fourteen glucose isotopologs from leaf extract were analysed by 13C NMR and interpreted in terms of their metabolic history.Tobacco plants grown in vitro were supplied with a mixture of [U-13C6]glucose and unlabelled sucrose via the root system. After 20 days, leaves were harvested and extracted with water. Glucose was isolated from the extract and was analysed by 13C NMR spectroscopy. All 13C signals appeared as complex multiplets due to 13C–13C coupling. The abundance of 21 isotopologous glucose species was determined from the 13C NMR signal integrals by numerical deconvolution using a genetic algorithm. The relative fractions of specific isotopologs in the overall excess of 13C-labelled specimens establish flux contributions via glycolysis/glucogenesis, pentose phosphate pathway, citric acid cycle and Calvin cycle including 13CO2 refixation. The fluxes were modelled and reconstructed in silico by a novel rule-based approach yielding the contributions of circular pathways and the degree of multiple cycling events. The data indicate that the vast majority of the proffered [U-13C6]glucose molecules had been modified by catabolism and subsequent glucogenesis from catabolic fragments, predominantly via passage through the citric acid cycle and the pentose phosphate pathway.
Keywords: Nicotiana tabacum; Metabolism; Carbon flux; NMR; Isotopolog; In silico modelling;

Variations in usnic acid concentrations in four widely separated populations of the lichen Flavocetraria nivalis are modestly correlated with time of season, as measured by the proximity in time to nearest summer solstice.The widespread secondary metabolite usnic acid, a dibenzofuran derivative, is the principal acetone-soluble compound in the lichen Flavocetraria nivalis. Seasonal variation in concentrations were studied in four populations of this lichen, three from Arctic–alpine habitats in the Northern Hemisphere, and one from Patagonian heathland in the Southern Hemisphere. Usnic acid is produced in large amounts, making up between 4% and 8% of thallus dry weight. Large seasonal variation is seen, with a trend towards peak levels in late spring and early summer, and generally low levels during autumn and winter. However, at an Arctic steppe in Central West Greenland, remarkably high levels were also detected during late autumn and early winter. Comparisons with environmental data using model selection procedures show that usnic acid levels of three of the populations are positively correlated with time of season, as measured by the proximity in time to nearest summer solstice, solar radiation levels, and temperature conditions. All these three variables are intercorrelated, thus indicating the same overall trend. For the three driest sites, precipitation rates are included in the models that best explain the variation in usnic acid. However, the explanatory powers of the models are generally low, partly due to high variation between thalli growing together and sampled at the same time. This is the first attempt to compare statistically seasonal variation in usnic acid concentrations and environmental variables, and thus also the first time it is shown that the concentration in various populations of the same lichen species shows different types of correlation with seasonal climatic changes.
Keywords: Flavocetraria nivalis; Lichenized ascomycetes; Seasonal variation; Secondary metabolism; Dibenzofurans; Usnic acid; Solar radiation; Ultraviolet radiation; AIC;

Encoded 3D structures of sesquiterpene lactones of three tribes of the family Asteraceae were projected into self-organizing maps allowing the prediction of occurrence of structures in taxa.This work describes an application of artificial neural networks on a small data set of sesquiterpene lactones (STLs) of three tribes of the family Asteraceae. Structurally different types of representative STLs from seven subtribes of the tribes Eupatorieae, Heliantheae and Vernonieae were selected as input data for self-organizing neural networks. Encoding the 3D molecular structures of STLs and their projection onto Kohonen maps allowed the classification of Asteraceae into tribes and subtribes. This approach allowed the evaluation of structural similarities among different sets of 3D structures of sesquiterpene lactones and their correlation with the current taxonomic classification of the family. Predictions of the occurrence of STLs from a plant species according to the taxa they belong to were also performed by the networks. The methodology used in this work can be applied to chemosystematic or chemotaxonomic studies of Asteraceae.
Keywords: Asteraceae; Chemoinformatics; Chemosystematics; Chemotaxonomy; Kohonen networks; Sesquiterpene lactones; 3D structures; Taxonomic markers;

Anti-protozoal and plasmodial FabI enzyme inhibiting metabolites of Scrophularia lepidota roots by Deniz Tasdemir; Nadide Deniz Güner; Remo Perozzo; Reto Brun; Ali A. Dönmez; Ihsan Çalıs; Peter Rüedi (355-362).
Nine iridoid glycosides, two of which (8, 9) are new, an iridoid-related aglycone (10) and a phenylethanoid glycoside, angoroside C (11), were isolated from the roots of S. lepidota. Compound 9 showed remarkable leishmanicidal activity (IC50 6.1 μg/ml), while 10 exhibited anti-malarial (40.6 g/ml) and plasmodial FabI inhibitory potential (IC50 100 μg/ml). 10 is the second anti-malarial natural product targeting the FabI enzyme of Plasmodium falciparum.The ethanolic root extract of Scrophularia lepidota, an endemic plant of the Turkish flora, has been investigated for its anti-protozoal and inhibitory effect towards plasmodial enoyl-ACP reductase (FabI), a key enzyme of fatty acid biosynthesis in Plasmodium falciparum. Chromatographic separation of the extract yielded 10 iridoids (110), two of which are new, and a known phenylethanoid glycoside (11). The structures of the new compounds were determined as 3,4-dihydro-methylcatalpol (8) and 6-O-[4″-O-trans-(3,4-dimethoxycinnamoyl)-α-l-rhamnopyranosyl]aucubin (scrolepidoside, 9) by spectroscopic means. The remaining metabolites were characterized as catalpol (1), 6-O-methylcatalpol (2), aucubin (3), 6-O-α-l-rhamnopyranosyl-aucubin (sinuatol, 4), 6-O-β-d-xylopyranosylaucubin (5), ajugol (6), ajugoside (7), an iridoid-related aglycone (10) and angoroside C (11). Nine isolates were active against Leishmania donovani, with the new compound 9 being most potent (IC50 6.1 μg/ml). Except for 4, all pure compounds revealed some trypanocidal potential against Trypanosoma brucei rhodesiense (IC50 values 29.3–73.0 μg/ml). Only compound 10 showed moderate anti-plasmodial (IC50 40.6 μg/ml) and FabI enzyme inhibitory activity (IC50 100 μg/ml). 10 is the second natural product inhibiting the fatty acid biosynthesis of Plasmodium falciparum.
Keywords: Scrophularia lepidota; Scrophulariaceae; Iridoid; Phenylethanoid glycoside; Plasmodium; Trypanosoma; Leishmania; Enoyl-acyl carrier protein reductase (FabI);

The isolation of two dehydrotriferulic acids from maize bran fiber demonstrates that ferulate trimers contribute to a strong network within the plant cell wall. These trimers do not contain a 5–5-coupled dimeric unit, hinting that more than two polysaccharide chains may be coupled by ferulate oligomers.Two new dehydrotriferulic acids were isolated from saponified maize bran insoluble fiber using Sephadex LH-20 chromatography followed by semi-preparative RP-HPLC. Based on UV-spectroscopy, mass spectroscopy and one- and two-dimensional NMR experiments, the structures were identified as 8–O–4,8–O–4-dehydrotriferulic acid and 8–8(cyclic),8–O–4-dehydrotriferulic acid. Which of the possible phenols in the initially formed 8–8-dehydrodiferulate was etherified by 4–O–8-coupling with ferulate has been unambiguously elucidated. The ferulate dehydrotrimers which give rise to these dehydrotriferulic acids following saponification are presumed, like the dehydrodiferulates, to cross-link polysaccharides. Neither dehydrotriferulic acid described here involves a 5–5-dehydrodiferulic acid unit; only the 5–5-dehydrodimer may be formed intramolecularly. However, whether dehydrotriferulates are capable of cross-linking more than two polysaccharide chains remains open. Although the levels of the isolated ferulate dehydrotrimers are lower than those of the ferulate dehydrodimers, the isolation now of three different dehydrotriferulates indicates that trimers contribute to a strong network cross-linking plant cell wall polysaccharides.
Keywords: Zea mays L.; Gramineae; Maize bran; Cell wall cross-linking; Triferulic acid; Triferulate; Ferulic acid; Ferulate; Arabinoxylans; Dietary fibre;

Alkaloids from Nerine filifolia by Jerald J. Nair; William E. Campbell; Reto Brun; Francesc Viladomat; Carles Codina; Jaume Bastida (373-382).
The alkaloids N-demethylbelladine, 6α-methoxybuphanidrine and filifoline are described for the first time from bulbs of Nerine filifolia (Amaryllidaceae). The nicotinate ester in filifoline was observed to reverse molecular ellipticity as expressed in the shape of the CD curve.The novel compounds N-demethylbelladine, 6α-methoxybuphanidrine and filifoline, in addition to five known alkaloids, and phenol have been isolated from fresh bulbs of Nerine filifolia (Amaryllidaceae). The structure and stereochemistry of the compounds were determined by physical and spectroscopic methods, including 1D and 2D NMR and mass spectroscopic techniques. An unusual circular dichroism response from filifoline has required a semi-synthetic derivatisation strategy towards key C-11endo analogues of the β-crinane representative ambelline in which the nature of substituents was observed to have a profound effect on molecular ellipticity. Filifoline was not cytotoxic to myoblast (L6) cells and exhibited no anti-protozoal activity in an in vitro screen against four different parasitic protozoa.
Keywords: Nerine filifolia; Amaryllidaceae; Bulbs; Alkaloids; Phenol; Belladine; N-Demethylbelladine; 6α-Hydroxybuphanidrine; 6α-Methoxybuphanidrine; Filifoline; Ambelline; 11-O-Acetylambelline; Undulatine; Circular dichroism;